Mechanical stability and differentially conserved physical-chemical properties of titin Ig-domains

Abstract

The mechanisms that determine mechanical stabilities of protein folds remain elusive. Our understanding of these mechanisms is vital to both bioengineering efforts and to the better understanding and eventual treatment of pathogenic mutations affecting mechanically important proteins such as titin. We present a new approach to analyze data from single-molecule force spectroscopy for different domains of the giant muscle protein titin. The region of titin found in the I-band of a sarcomere is composed of about 40 Ig-domains and is exposed to force under normal physiological conditions and connects the free-hanging ends of the myosin filaments to the Z-disc. Recent single-molecule force spectroscopy data show a mechanical hierarchy in the I-band domains. Domains near the C-terminus in this region unfold at forces two to three times greater than domains near the beginning of the I-band. Though all of these Ig-domains are thought to share a fold and topology common to members of the Ig-like fold family, the sequences of neighboring domains vary greatly with an average sequence identity of only 25%. We examine in this study the relation of these unique mechanical stabilities of each I-band Ig domain to specific, conserved physical-chemical properties of amino acid sequences in related Ig domains. We find that the sequences of each individual titin Ig domain are very highly conserved, with an average sequence identity of 79% across species that are divergent as humans, chickens, and zebra fish. This indicates that the mechanical properties of each domain are well conserved and tailored to its unique position in the titin molecule. We used the PCPMer software to determine the conservation of amino acid properties in titin Ig domains grouped by unfolding forces into "strong" and "weak" families. We found two motifs unique to each family that may have some role in determining the mechanical properties of these Ig domains. A detailed statistical analysis of properties of individual residues revealed several positions that displayed differentially conserved properties in strong and weak families. In contrast to previous studies, we find evidence that suggests that the mechanical stability of Ig domains is determined by several residues scattered across the beta-sandwich fold, and force sensitive residues are not only confined to the A'-G region.

Figure 1. Location and architecture of titin, and the structure of an Ig domain

A) Single titin molecules (black lines) span half the length of the sarcomere. B) The domain architecture of the I-band of the N2-B titin isoform is composed almost entirely of Ig-like domains and is exposed to tensile forces in vivo. The domains used in our analysis are highlighted in cyan (I1, I4, I5, I27, I28, I32, I34). C) The structure of I27 (pdb code 1TIT21) is given as an example of the beta-sandwich, Ig-like domain repeated approximately 40 times in the I-band of human cardiac N2-B titin. The repeats occur head-to-tail producing an extended chain similar to beads on a necklace. The topology of the domains lends itself to mechanical resistance when its N and C termini are pulled in opposite directions.

Figure 2. Characterization of the mechanical stabilities of titin I-band domains

A) A force-extension of a polyprotein consisting of multiple repeats of identical I27 Ig domains. The lines correspond to fits to the worm-like chain equation. B) Histogram of the measured unfolding forces for this polyprotein; the mean value is 220+/-20pN (n=115). C) Plot of the unfolding force versus the location of different titin Ig domains in the I-band of human cardiac N2-B. Seven I-band Ig domains have been characterized to reveal a mechanical hierarchy in the I-band in which the proximal domains unfold at significantly lower forces when compared to distal domains (Data from Li et. al. 2002 and 2003). The mean unfolding force for both groups are shown as red (strong family) and blue (weak family) lines.

Figure 3. Dot plot of the sequence identities of cardiac titin I-band Ig domains

Sequence identities of 10% or less begin at black and proceed in a smooth gradient to bright red then yellow as the identities approach 40%. Identities of 40% and over are emphasized by a shift through white to blue at identities of 65%. The 100% identity diagonal squares are colored a pale cyan. At least three self-similar groupings become obvious from this plot. The groups consist of domains I2 to I13 (I), domains I14 to I20 (II), and domains I23 to I39 (III). Domains I1, I21, and I22 each seem quite dissimilar to other domains and each other. Group III contains a large number of high sequence identity pairs and may be an example of gene duplication.

Figure 4. Sequence alignments of seven I-band Ig domains

for which the unfolding force is known. The domains are grouped by rupture force into two families: A) a strong family consisting of I27, I28, I32, and I34 and B) a weak family consisting of I1, I4, and I5.

Figure 5. Best scoring window of motifs in each sequence

This figure illustrates the conservation of some motifs across families and the uniqueness of others. A) Motifs of the weak family are shown in unique colors (w1: red, w2: orange, w3: yellow, w4: green, w5: blue, w6: violet) highlighting their best matching window for each human sequence in the weak and strong families. B) Motifs of the strong family (s1: red, s2: orange, s3: yellow, s4: green, s5: blue, s6: violet) highlight their best matching window in each of the human sequences in both families. Here we can easily see the conservation of motifs w2, w4, w5 and s3, s5, and s6 across both families.

Figure 6. Comparison of the scores for the six weak and strong motifs to find unique motifs

In the top part all six weak motifs are matched against all sequences and scores are given above the zero line for sequences in the weak family (averaged as described in the Methods part) and below the zero line for sequences in the strong family. Each weak motif is highlighted in a different color in the representative human sequence of I1 and the colors of the score bars are characteristic for each motif. The middle part of the figure gives the secondary structure of the Ig domains with the representative human sequence of I1 for the weak family and I27 for the strong family. The lower part represents the scores of the six motifs derived from the strong family matched individually through the sequences of the strong family and weak family with scores given as in the top portion.

Figure 7. Positions found to be significantly different between the weak and strong families

Residues with significant property shifts from the weak to strong families are displayed on the structure of I27 (pdb code 1TIT21) with a small C_α sphere and large C_β sphere. A) The first vector E1 correlates well with descriptors of hydrophilicity/hydrophobicity. Red colored residues indicate a shift from hydrophobic values toward hydrophilic in the strong family, while blue indicate the reverse shift. There are more hydrophobic shifts with many of these in loops or extended regions of the domain. B) The second vector E2 describes the size of the side-chains. Red colored residues indicate a shift from larger side-chain values toward smaller ones in the strong family, while blue indicate the reverse shift. More residues increase in the size of side-chains when we compare sequences from the strong family versus the weak family.